In many industries, particularly the alimentary, pharmaceutical and electronic industries, as well as in hospitals, it is essential to be able to evaluate the degree of contamination of a number of liquids by microorganisms, i.e., bacteria, yeasts or molds.
To effect such an evaluation, it is preferable to use membrane filtration techniques as opposed to the process of growing a culture medium in a Petri dish which only permits analysis of samples of a small volume.
The membrane filtration method consists of filtering the sample through a sterile membrane placed on a porous holder onto which a funnel for receiving the sample is sealingly fixed. Prior to filtration, both the holder and the funnel are decontaminated.
After filtration, which is generally effected through a vacuum flask, and rinsing to eliminate bacteriostatic agents possibly present, the membrane is removed with sterile forceps and deposited onto a gelatinous culture medium contained in a Petri dish. The Petri dish is then incubated at a suitable temperature for the time necessary for the microorganisms to be able to develop and multiply sufficiently to form colonies visible to the naked eye to permit them to be identified and counted.
This known membrane filtration method permits evaluation of even slightly contaminated samples because the microorganisms are concentrated on the membrane, and it is possible to filter a significant volume of sample to collect a sufficient number of microorganisms. However, this method presents certain disadvantages. First, it must be carried out in a major laboratory involving many sterilized utensils and the use of specialized personnel because of the complicated manipulations required. Also the rinsing of the membrane by a sterile rinsing liquid does not always reach the peripheral zones where the membrane is clamped between its holder and the funnel bottom. Thus any bacteriostatic agents possibly present may be retained in such zones and may migrate upon incubation thereby hampering multiplication of the microorganisms to be counted.
Moreover, care must be exercised in placing the membrane filter upon the culture medium in the Petri dish to avoid entrapping any air bubbles between the culture medium and the membrane which would prevent contact between the culture medium and a portion of the surface of the membrane thereby opposing diffusion of the culture medium, and inhibiting microorganism development.
Finally, the use of vacuum filtration does not allow verification that the filter membrane, the pore diameter of which was calculated to retain a certain type of microorganism (e.g., 0.45 micrometers for bacteria) remained intact during the manipulations to which it was submitted.
To remedy some of these inconveniences, a filtration procedure has been proposed consisting of using a sterile box with circular elements nested into one another and a removable cover. This box includes an inlet and an outlet disposed on both sides of a holder on which an absorbent pad and a filtration membrane lie, clamped at the periphery to the holder by one of the circular elements. After filtration, which is generally accomplished through aspiration of the sample that passes successively through the membrane and then the absorbent pad, a culture medium is introduced through the outlet, that is, counter-currently to normal operation, to saturate the absorbent pad. Once the outlet has been obstructed, the box can be directly brought to the incubator to permit the collected microorganisms to develop.
Although this membrane filtration method using a sterile box avoids membrane manipulation by an operator, and permits the samples to be directly taken on the site, it has, however, serious drawbacks. First, the membrane diameter increases when it becomes wetted and since the membrane and the absorbent pad, which are clamped at their periphery to the sterile box are kept dry prior to use, upon filtration of the sample the wetting of the pad and membrane may cause the membrane filter to part from the absorbent pad. This prevents contact between the membrane and the pad saturated with the culture medium, and thus disturbs development of the microorganism colonies upon incubation.
In the same manner as in the preceding techniques, it is not possible to prevent, in spite of membrane rinsing, migration of bacteriostatic agents retained in the peripheral zone of the membrane to its center where these agents could inhibit development of the microorganisms to be counted. Similarly, as the absorbent pad which is wetted upon filtration of the sample can also contain growth inhibiting elements, the culture medium with which the absorbent pad is subsequently saturated can become proportionately diluted and possibly modified by such elements. Finally, introduction of the culture medium in a counter-current direction on the absorbent pad may cause the membrane to be lifted and moved away from the pad thereby forcibly causing disturbance in the growth of the microorganisms to be counted.
A third filtration method consists of using a sampler of the "dipstick" type which as indicated by its name is in form of a dipstick and comprises a membrane filter covering an absorbent pad impregnated with a culture medium dried by lyophilization. By dipping this sampler into the liquid to be analyzed, there is pumped by capillary action through the membrane a certain volume of liquid which saturates the lyophilized culture medium by solubilizing it. The impregnated dipstick can be directly brought to incubation.
This third filtration procedure is however of a limited use since it can hardly ever be used for analyzing water. The analyzed sample volume is in the order of a few cubic centimeters (cm.sup.3) only and the reconstitution of a culture medium involves supplementary risks as compared to the techniques discussed above.